Search

Bax is a proapoptotic protein that is required for programmed cell death (PCD) of many neuronal populations. Here we show that, during an early period of retinal PCD and in naturally occurring sensory and motor neuron (MN) death in the spinal cord, Bax delivery results in enhanced death of these neural populations. In contrast, Bax overexpression fails to enhance an early phase of MN death that occurs in the cervical spinal cord, although overexpressed Bax appears to be activated in dying MNs. Bax overexpression does not also affect the survival of immature neurons prior to the PCD period. Taken together, these data provide the first in vivo evidence suggesting that Bax appears to act selectively as an executioner only in neurons undergoing PCD. Furthermore, although Bax appears to mediate the execution pathway for PCD, the effect of Bax overexpression on susceptibility to death differs between different neuronal populations.Cell Death and Differentiation (2006) 13, 435–445. doi:10.1038/sj.cdd.4401760; published online 9 September 2005 [ABSTRACT FROM AUTHOR]

Mosquitoes obtain large amounts of blood from hosts in a short period of time. To efficiently obtain high-quality blood without being noticed by the host, mosquitoes sense external factors such as the taste of the host blood and the surrounding environment, and integrate these signals with their own internal information to determine whether to initiate blood feeding and how long to continue feeding. With the development of gene editing and behavior monitoring techniques, the factors that control blood feeding are being identified. Elucidating the factors that contribute to blood feeding is expected to provide new ideas for artificially controlling blood feeding, which has often been overlooked behind host attraction mechanisms. Furthermore, understanding salivary components, mechanisms controlling satiety in feeding, and differences between sugar feeding and blood feeding would help us understand how some mosquitoes have adopted and developed blood feeding over the course of evolution., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Dietary protein intake modulates growth, reproduction, and longevity by stimulating amino acid (AA)-sensing pathways. Essential AAs are often considered as limiting nutrients during protein scarcity, and the role of dietary non-essential AAs (NEAAs) is less explored. Although tyrosine has been reported to be crucial for sensing protein restriction in Drosophila larvae, its effect on adult physiology and longevity remains unclear. Here, using a synthetic diet, we perform a systematic investigation of the effect of single NEAA deprivation on nutrient-sensing pathways, reproductive ability, starvation resistance, feeding behavior, and life span in adult female flies. Specifically, dietary tyrosine deprivation decreases internal tyrosine levels and fecundity, influences AA-sensing machineries, and extends life span. These nutritional responses are not observed under higher total AA intake or in infertile female flies, suggesting a context-dependent influence of dietary tyrosine. Our findings highlight the unique role of tyrosine as a potentially limiting nutrient, underscoring its value for dietary interventions aimed at enhancing health span.

Female mosquitoes engage in blood feeding from their hosts to facilitate egg maturation but cease feeding once a sufficient blood meal has been acquired. Abdominal distention has been proposed as a contributing factor; however, it has also been suggested that there are chemical controls. In this study, we focus on negative chemical regulators of blood feeding, particularly those present in the host blood. Serum derived from animal blood inhibits the feeding of ATP, a phagostimulant of blood feeding in Aedes aegypti. Fibrinopeptide A (FPA), a 16-amino acid peptide cleaved from fibrinogen during blood coagulation, serves as an inhibitory factor in the serum. Our findings suggest that blood-feeding arrest in female mosquitoes is triggered by the detection of FPA in the host blood, which increases as blood coagulation proceeds in the mosquito's midgut, highlighting the role of host-derived substances as negative regulators of mosquito behavior., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Atg8 paralogs, consisting of LC3A/B/C and GBRP/GBRPL1/GATE16, function in canonical autophagy; however, their function is controversial because of functional redundancy. In innate immunity, xenophagy and non-canonical single membranous autophagy called "conjugation of Atg8s to single membranes" (CASM) eliminate bacteria in various cells. Previously, we reported that intracellular Streptococcus pneumoniae can induce unique hierarchical autophagy comprised of CASM induction, shedding, and subsequent xenophagy. However, the molecular mechanisms underlying these processes and the biological significance of transient CASM induction remain unknown. Herein, we profile the relationship between Atg8s, autophagy receptors, poly-ubiquitin, and Atg4 paralogs during pneumococcal infection to understand the driving principles of hierarchical autophagy and find that GATE16 and GBRP sequentially play a pivotal role in CASM shedding and subsequent xenophagy induction, respectively, and LC3A and GBRPL1 are involved in CASM/xenophagy induction. Moreover, we reveal ingenious bacterial tactics to gain intracellular survival niches by manipulating CASM-xenophagy progression by generating intracellular pneumococci-derived H 2 O 2 ., Competing Interests: Declaration of interests Authors declare that they have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective A short sleep duration is associated with non-alcoholic fatty liver disease (NAFLD). However, the causal relationship between a short sleep duration and the onset of NAFLD remains unknown because of the lack of any longitudinal studies. Therefore, we evaluated the association between sleep duration and the onset of NAFLD. Methods We evaluated health checkup data for 1,862 NAFLD-free Japanese adults aged 33-86 years at baseline and followed those individuals for a median of 41 months. Hepatic steatosis was examined using ultrasonography (US). The Cox proportional hazards model was used to evaluate the association between sleep duration and NAFLD onset. Results Among the 1,862 participants, 483 (25.9%) developed NAFLD. The proportion of women who developed NAFLD was the highest in the group with a sleep duration of <6 hours and lowest in the group with a sleep duration of 7 to <8 hours. The multivariable-adjusted hazard ratio (95% confidence interval) for the onset of NAFLD in women with a sleep duration <6 hours compared with those with a sleep duration of 7 to <8 hours was 1.55 (1.09-2.20; p=0.02). Conclusions In women, a short sleep duration was independently associated with the onset of NAFLD, thus suggesting that an adequate sleep duration can be a promising preventive factor for the onset of NAFLD in women.

Our research activities would be better served if they were communicated in a manner that is openly accessible to the public and all researchers. The research we share is often limited to representative data included in research papers-science would be much more efficient if all reproducible research data were shared alongside detailed methods and protocols, in the paradigm called Open Science. On the other hand, one primary function of research journals is to select manuscripts of good quality, verify the authenticity of the data and its impact, and deliver to the appropriate audience for critical evaluation and verification. In the current paradigm, where publication in a subset of journals is intimately linked to research evaluation, a hypercompetitive "market" has emerged where authors compete to access a limited number of top-tier journals, leading to high rejection rates. Competition among publishers and scientific journals for market dominance resulted in an increase in both the number of journals and the cost of publishing and accessing scientific papers. Here we summarize the current problems and potential solutions from the development of AI technology discussed in the seminar at the 46th Annual Meeting of the Molecular Biology Society of Japan., (© 2024 The Authors. Genes to Cells published by Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Pneumolysin (Ply) is an indispensable cholesterol-dependent cytolysin for pneumococcal infection. Although Ply-induced disruption of pneumococci-containing endosomal vesicles is a prerequisite for the evasion of endolysosomal bacterial clearance, its potent activity can be a double-edged sword, having a detrimental effect on bacterial survivability by inducing severe endosomal disruption, bactericidal autophagy, and scaffold epithelial cell death. Thus, Ply activity must be maintained at optimal levels. We develop a highly sensitive assay to monitor endosomal disruption using NanoBiT-Nanobody, which shows that the pneumococcal sialidase NanA can fine-tune Ply activity by trimming sialic acid from cell-membrane-bound glycans. In addition, oseltamivir, an influenza A virus sialidase inhibitor, promotes Ply-induced endosomal disruption and cytotoxicity by inhibiting NanA activity in vitro and greater tissue damage and bacterial clearance in vivo. Our findings provide a foundation for innovative therapeutic strategies for severe pneumococcal infections by exploiting the duality of Ply activity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The frequency of cleft lip with/without palate (CL/P) in the Mongolian population is approximately 1 in 1314 live births. This research aims to disseminate information about this congenital disability to the public to better understand CL/P, and people's fissures, and review administrative measures, as there is a lack of research in this area. A questionnaire survey was conducted using Google Forms, with 1000 Mongolian participants. Most participants (86.7%) said they had knowledge of the word, whereas 86.2% said they had knowledge of the condition. Most participants' answers were question-related disadvantages of CL/P patients, including statements such as "It's uncomfortable in human relationships" and "It makes an uncomfortable impression on the person you meet the first time." The results of this study revealed that most Mongolians were aware of CL/P and are concerned about patients. However, the causes of CL/P in the general population remain unknown, and further research is needed in this area., (© 2024 Japanese Teratology Society.)

Gel electrophoresis, a transport technology, is one of the most widely used experimental methods in biochemical and pharmaceutical research and development. Transport technologies are used to determine hydrodynamic or electrophoretic properties of macromolecules. Gel electrophoresis is a zone technology, where a small volume of sample is applied to a large separation gel matrix. In contrast, a seldom-used electrophoresis technology is moving boundary electrophoresis, where the sample is present throughout the separation phase or gel matrix. While the zone method gives peaks of separating macromolecular solutes, the moving boundary method gives a boundary between solute-free and solute-containing phases. We will review electrophoresis as a transport technology of zone and moving boundary methods and describe its principles and applications., (© 2023. European Biophysical Societies' Association.)

Heat is one of the critical cues female mosquitoes use to find vertebrate hosts, particularly in the final step of attraction (touching down on hosts) before the initiation of blood-sucking. To prevent vector-borne diseases such as malaria and dengue fever, which are transmitted via mosquito blood-sucking, it is essential to understand the dynamics and mechanisms of the heat-seeking behavior of mosquitoes. An automated device to quantify CO 2 -activated heat-seeking behavior with continuous monitoring for up to a week was established. The device is based on the infrared beam break method; it can simultaneously monitor three independent mosquito behaviors (touchdown on a heated target, feeding, and locomotion activity) by using several pairs of infrared laser sensors. This protocol describes brief instructions for the device's construction, directions for use, and possible issues and solutions., (© 2023 Cold Spring Harbor Laboratory Press.)

Mosquitoes are vectors of various deadly infectious diseases, including malaria and dengue fever. Because these pathogens are transmitted by mosquito blood-feeding behavior, it is important to understand how mosquitoes are attracted to their hosts and how they feed on blood. The simplest method is to observe their behavior with the naked eye or video recording. Furthermore, various devices have been invented to assay mosquito behavior, such as olfactometers. Although each method has distinctive advantages, all have disadvantages, such as limitations in the number of individuals that can be assayed at one time and in observable durations, objective quantification methods, and others. To solve these problems, we have developed an automated device to quantify the carbon dioxide-activated heat-seeking behavior of Anopheles stephensi and Aedes aegypti with continuous monitoring for up to 1 week. This device, which is detailed in an accompanying protocol, can be applied to search for substances and molecules that affect heat-seeking behavior. It may also be applicable to other hematophagous insects., (© 2023 Cold Spring Harbor Laboratory Press.)

This study aims to identify potential variants in the TP63-IRF6 pathway and GREM1 for the etiology of non-syndromic orofacial cleft (NSOFC) among the Vietnamese population. By collecting 527 case-parent trios and 527 control samples, we conducted a stratified analysis based on different NSOFC phenotypes, using allelic, dominant, recessive and over-dominant models for case-control analyses, and family-based association tests for case-parent trios. Haplotype and linkage disequilibrium analyses were also conducted. IRF6 rs2235375 showed a significant association with an increased risk for non-syndromic cleft lip and palate (NSCLP) and cleft lip with or without cleft palate (NSCL/P) in the G allele, with p allele values of 0.0018 and 0.0003, respectively. Due to the recessive model ( p = 0.0011) for the NSCL/P group, the reduced frequency of the GG genotype of rs2235375 was associated with a protective effect against NSCL/P. Additionally, offspring who inherited the G allele at rs2235375 had a 1.34-fold increased risk of NSCL/P compared to the C allele holders. IRF6 rs846810 and a G-G haplotype at rs2235375-rs846810 of IRF6 impacted NSCL/P, with p -values of 0.0015 and 0.0003, respectively. In conclusion, our study provided additional evidence for the association of IRF6 rs2235375 with NSCLP and NSCL/P. We also identified IRF6 rs846810 as a novel marker associated with NSCL/P, and haplotypes G-G and C-A at rs2235375-rs846810 of IRF6 associated with NSOFC.

Objectives: Endoscopic submucosal dissection (ESD) enables en bloc resection of colorectal neoplasms, but occasionally results in positive horizontal margins (HMs). However, the site of the resected specimen that tends to be positive for HM has not been investigated. We aimed to clarify the characteristics associated with HMs in lesions resected en bloc with ESD., Methods: Patients with colorectal neoplasms who underwent en bloc resection with ESD were included in this study. The patients were divided into negative HMs (HM0) and positive or indeterminate HMs (HM1) groups. The characteristics associated with HM1 resection were investigated. In addition, the local recurrence rate during endoscopic follow-up for >6 months after ESD was observed., Results: In total, 201 lesions were analyzed in 189 patients (HM0, 189 lesions; HM1, 12 lesions). The HM1 group had a significantly larger median lesion diameter (25 vs. 55 mm; p  < 0.001) and more lesions with >50% circumference than did the HM0 group ( p  < 0.001). Furthermore, the prevalence of severe fibrosis was significantly higher in the HM1 group than in the HM0 group ( p  < 0.001). Positive horizontal sites of the resected specimens were more frequent at the oral and anal sites than at the lateral sites. No local recurrences were observed in either group., Conclusions: The characteristics associated with HM1 depended on lesion size, particularly lesions with >50% circumference, and submucosal fibrosis., Competing Interests: Authors declare no conflicts of interest for this article., (© 2023 The Authors. DEN Open published by John Wiley & Sons Australia, Ltd on behalf of Japan Gastroenterological Endoscopy Society.)

A new protocol for conducting two-dimensional (2D) electrophoresis was developed by combining the recently developed agarose native gel electrophoresis with either vertical sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) or flat SDS agarose gel electrophoresis. Our innovative technique utilizes His/MES buffer (pH 6.1) during the first-dimensional (1D) agarose native gel electrophoresis, which allows for the simultaneous and clear visualization of basic and acidic proteins in their native states or complex structures. Our agarose gel electrophoresis is a true native electrophoresis, unlike blue native-PAGE, which relies on the intrinsic charged states of the proteins and their complexes without the need for dye binding. In the 2D, the gel strip from the 1D agarose gel electrophoresis is soaked in SDS and placed on top of the vertical SDS-PAGE gels or the edge of the flat SDS-MetaPhor high-resolution agarose gels. This allows for customized operation using a single electrophoresis device at a low cost. This technique has been successfully applied to analyze various proteins, including five model proteins (BSA, factor Xa, ovotransferrin, IgG, and lysozyme), monoclonal antibodies with slightly different isoelectric points, polyclonal antibodies, and antigen-antibody complexes, as well as complex proteins such as IgM pentamer and β-galactosidase tetramer. Our protocol can be completed within a day, taking approximately 5-6 h, and can be expanded further into Western blot analysis, mass spectrometry analysis, and other analytical methods., (© 2023 Wiley-VCH GmbH.)

Background: The optic tectum is the main visual processor of nonmammalian vertebrates and relays visual information from the eye to the telencephalon via the tectofugal pathway. In the development of the avian optic tectum, while the multipolar neurons are arranged by tangential migration, the behavior of individual cells in tangential migration, neural differentiation, and cell fate remain unclear. Here, we pursued the transition of tangentially migrating cells and their involvement in visual circuit formation during chick development., Results: After tangential movement along the axons, the migrating cells relocated to the upper layers and turned back upon differentiation toward the multipolar neurons. The multipolar neurons are destined to differentiate into the stratum griseum central (SGC) neurons with the large dendritic field, which form the tectorotundal projection. Trans-synaptic virus labeling demonstrated that the tangentially migrating cells eventually participate in the tectofugal visual pathway., Conclusions: These results indicate that tangential migration is a crucial process in the formation of the tectofugal visual pathway during the development of the optic tectum., (© 2023 American Association for Anatomy.)

Currently, purification of antibodies is mainly carried out using a platform technology composed primarily of Protein A chromatography as a capture step, regardless of the scale. However, Protein A chromatography has a number of drawbacks, which are summarized in this review. As an alternative, we propose a simple small-scale purification protocol without Protein A that uses novel agarose native gel electrophoresis and protein extraction. For large-scale antibody purification, we suggest mixed-mode chromatography that can in part mimic the properties of Protein A resin, focusing on 4-Mercapto-ethyl-pyridine (MEP) column chromatography.

The nucleoprotein (NP) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is abundantly expressed during infection, making it a diagnostic target protein. We analyzed the structure of the NP in solution using a recombinant protein produced in E. coli. A codon-optimized Profinity eXact™-tagged NP cDNA was cloned into pET-3d vector and transformed into E. coli T7 Express. The recombinant protein was first purified via chromatographic step using an affinity tag-based system that was followed by tag cleavage with sodium fluoride, resulting in proteolytic removal of the N-terminal tag sequence. The digested sample was then loaded directly onto a size exclusion chromatography run in the presence of L-Arg-HCl, resulting in removal of host nucleic acids and endotoxin. The molecular mass of the main NP fraction was determined by mass photometry as a dimeric form of NP, consistent with the blue native PAGE results. Interestingly, analysis of the purified NP by our newly developed agarose native gel electrophoresis revealed that it behaved like an acidic protein at low concentration despite its alkaline isoelectric point (theoretical pI = 10) and displayed a unique character of concentration-dependent charge and shape changes. This study should shed light into the behavior of NP in the viral life cycle., (Copyright © 2022 Elsevier Inc. All rights reserved.)

An attempt was made to specifically stain unfolded proteins on agarose native gels. SYPRO Orange is routinely used to detect unfolded protein in differential scanning fluorimetry, which is based on the enhanced fluorescence intensity upon binding to the unfolded protein. We demonstrated that this dye barely bound to the native proteins, resulting in no or faint staining of the native bands, but bound to and stained the unfolded proteins, on agarose native gels. Using bovine serum albumin (BSA), it was shown that staining did not depend on whether BSA was thermally unfolded in the presence of SYPRO Orange or stained after electrophoresis. On the contrary, SYPRO Orange dye stained protein bands in the presence of sodium dodecylsulfate (SDS) due to incorporation of the dye into SDS micelles that bound to the unfolded proteins. This staining resulted in detection of new, intermediately unfolded structure of BSA during thermal unfolding. Such intermediate structure occurred at higher temperature in the presence of ATP., (Copyright © 2022 Elsevier Inc. All rights reserved.)

A commercially available bovine serum albumin (BSA) was examined by agarose native gel electrophoresis using two different agarose sources, UltraPure and MetaPhor agarose. While UltraPure agarose up to 5 % showed no clear separation of BSA oligomers, MetaPhor agarose clearly demonstrated oligomer bands above 4 %, indicating that the latter agarose has greater molecular sieving effects and is hence characterized to have high resolution for size differences, as probed by a greater slope of Ferguson plot. Physical properties are different between two agaroses. In general, UltraPure agarose has physical strength, while MetaPhor agarose is considerably fragile, but MetaPhor agarose solution is less viscous so that even 10 % gel can be made. Cause of oligomers was shown to be not associated with inter-chain disulfide bonds, but is due to association of native or native-like molecules., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In this study, we review the agarose native gel electrophoresis that separates proteins and macromolecular complexes in their native state and transfer of the separated proteins from the agarose gel to membranes by contact blotting which retains the native state of these structures. Green fluorescent protein showed functional state both on agarose gel and blotted membrane. Based on the combined procedures, we discovered conformation-specific monoclonal antibodies against PLXDC2 and SARS-CoV-2 spike protein.

We have developed a new Western blotting method of native proteins from agarose-based gel electrophoresis using a buffer at pH 6.1 containing basic histidine and acidic 2-( N- morpholino)ethanesulfonic acid. This gel electrophoresis successfully provided native structures for a variety of proteins and macromolecular complexes. This paper is focused on the Western blotting of native protein bands separated on agarose gels. Two blotting methods from agarose gel to PVDF membrane are introduced here, one by contact (diffusion) blotting and another by electroblotting after pre-treating the agarose gels with SDS. The contact blotting resulted in the transfer of native GFP, native human plexin domain containing protein 2 (PLXDC2) and native SARS-CoV-2 spike protein, which were detected by conformation-specific antibodies generated in-house.

The Vero cell line is an immortalized cell line established from kidney epithelial cells of the African green monkey. A variety of Vero sublines have been developed and can be classified into four major cell lineages. In this study, we determined the whole-genome sequence of Vero E6 (VERO C1008), which is one of the most widely used cell lines for the proliferation and isolation of severe acute respiratory syndrome coronaviruses (SARS-CoVs), and performed comparative analysis among Vero JCRB0111, Vero CCL-81, Vero 76, and Vero E6. Analysis of the copy number changes and loss of heterozygosity revealed that these four sublines share a large deletion and loss of heterozygosity on chromosome 12, which harbors type I interferon and CDKN2 gene clusters. We identified a substantial number of genetic differences among the sublines including single nucleotide variants, indels, and copy number variations. The spectrum of single nucleotide variants indicated a close genetic relationship between Vero JCRB0111 and Vero CCL-81, and between Vero 76 and Vero E6, and a considerable genetic gap between the former two and the latter two lines. In contrast, we confirmed the pattern of genomic integration sites of simian endogenous retroviral sequences, which was consistent among the sublines. We identified subline-specific/enriched loss of function and missense variants, which potentially contribute to the differences in response to viral infection among the Vero sublines. In particular, we identified four genes ( IL1RAP , TRIM25 , RB1CC1 , and ATG2A ) that contained missense variants specific or enriched in Vero E6. In addition, we found that V739I variants of ACE2, which functions as the receptor for SARS-CoVs, were heterozygous in Vero JCRB0111, Vero CCL-81, and Vero 76; however, Vero E6 harbored only the allele with isoleucine, resulting from the loss of one of the X chromosomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Konishi, Yamaji, Sakuma, Kasai, Endo, Kohara, Hanada and Osada.)

Background: Mosquito control is a crucial global issue for protecting the human community from mosquito-borne diseases. There is an urgent need for the development of selective and safe reagents for mosquito control. Flavonoids, a group of chemical substances with variable phenolic structures, such as daidzein, have been suggested as potential mosquito larvicides with less risk to the environment. However, the mode of mosquito larvicidal action of flavonoids has not been elucidated., Results: Here, we report that several flavonoids, including daidzein, inhibit the activity of glutathione S-transferase Noppera-bo (Nobo), an enzyme used for the biosynthesis of the insect steroid hormone ecdysone, in the yellow fever mosquito Aedes aegypti. The crystal structure of the Nobo protein of Ae. aegypti (AeNobo) complexed with the flavonoids and its molecular dynamics simulation revealed that Glu113 forms a hydrogen bond with the flavonoid inhibitors. Consistent with this observation, substitution of Glu113 with Ala drastically reduced the inhibitory activity of the flavonoids against AeNobo. Among the identified flavonoid-type inhibitors, desmethylglycitein (4',6,7-trihydroxyisoflavone) exhibited the highest inhibitory activity in vitro. Moreover, the inhibitory activities of the flavonoids correlated with the larvicidal activity, as desmethylglycitein suppressed Ae. aegypti larval development more efficiently than daidzein., Conclusion: Our study demonstrates the mode of action of flavonoids on the Ae. aegypti Nobo protein at the atomic, enzymatic, and organismal levels., (© 2022. The Author(s).)

Rupture of the basement membrane in fused palate tissue can cause the palate to separate after fusion in mice, leading to the development of cleft palate. Here, we further elucidate the mechanism of palatal separation after palatal fusion in 8-10-week-old ICR female mice. On day 12 of gestation, 40 μg/kg of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), sufficient to cause cleft palate in 100% of mice, was dissolved in 0.4 mL of olive oil containing toluene and administered as a single dose via a gastric tube. Fetal palatine frontal sections were observed by H&E staining, and epithelial cell adhesion factors, apoptosis, and cell proliferation were observed from the anterior to posterior palate. TUNEL-positive cells and Ki67-positive cells were observed around the posterior palatal dissection area of the TCDD-treated group. Moreover, in fetal mice exposed to TCDD, some fetuses exhibited cleft palate dehiscence during fusion. The results suggest that palatal dehiscence may be caused by abnormal cell proliferation in epithelial tissues, decreased intercellular adhesion, and inhibition of mesenchymal cell proliferation. By elucidating the mechanism of cleavage after palatal fusion, this research can contribute to establishing methods for the prevention of cleft palate development.

Electrophoresis is one of the most important analytical technologies for characterization of macromolecules and their interactions. Among them, native gel electrophoresis is used to analyze the macromolecules in the native structure. It differs in principle and information from those obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) or blue native polyacrylamide gel electrophoresis (BN-PAGE). SDS-PAGE is carried out in the presence of strong denaturant, SDS, while BN-PAGE is done in the presence of negatively charged dye, e.g., Coomassie brilliant blue, G-250. Here, we describe native gel electrophoresis using agarose gel and a buffer at pH 6.1 composed of histidine and 2-(N-morpholino) ethanesulfonic acid. First, a protocol for vertical and horizontal formats of agarose native gel electrophoresis is described followed by different staining procedures. Then, various examples obtained using the developed procedure will be shown to demonstrate how the technology can be applied to specific cases and the advantages or caveats of the present technology., (Copyright © 2022 Elsevier Inc. All rights reserved.)

We have been conducting a survey on the birth prevalence of orofacial clefts, including cleft lip with or without cleft palate and cleft palate, in the Tokai area in central Japan every year for 37 years. Along with the yearly trends in the birth prevalence of orofacial clefts in that area for the past 37 years, we discuss whether the artificial abortion rate of fetuses with orofacial clefts has increased through the improved performance of ultrasonic imaging equipment. We also compare the yearly trends in the birth prevalence of congenital anomalies, including orofacial clefts, in Japan with those in other countries or areas where artificial abortion due to birth defects is legally permitted, and discuss the impact of improved accuracy of ultrasound imaging on the rate of artificial termination of pregnancy. The fact that the birth prevalence of orofacial clefts has basically remained unchanged for more than 30 years, even with recent more detailed prenatal diagnosis based on the improvement of ultrasonic diagnostic equipment, has allowed us tentatively to conclude that prenatal diagnosis is not currently threatening the right to life of the fetuses with orofacial clefts., (© 2021 Japanese Teratology Society.)

Electrophoresis is one of the major techniques to analyze macromolecular structure and interaction. Its capability depends on the sensitivity and specificity of the staining methods. We have here examined silver staining of proteins and nucleic acids separated by agarose native gel electrophoresis. By comparing five commercial kits, we identified Silver Stain Plus from Bio-Rad most adequate, as it provided little background staining and reasonable band staining. One of the disadvantages of the Silver Stain Plus kit is its variable staining of glycoproteins as tested with several model samples, including hen egg white proteins, α1-acid glycoprotein and SARS-CoV-2 Spike protein. One of the advantages of silver staining is its ability to stain nucleic acids as demonstrated here for a model nucleic acid with two kits. It was then used to monitor the removal of nucleic acids from the affinity-purified maltose binding protein and monoclonal antibody. It also worked well on staining proteins on agarose gels prepared in the vertical mode, although preparation of the vertical agarose gels required technological modifications described in this report. With the silver staining method optimized here, it should be possible in the future to analyze biological samples that may be available in limited quantity., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The developmental hourglass model predicts that embryonic morphology is most conserved at the mid-embryonic stage and diverges at the early and late stages. To date, this model has been verified by examining the anatomical features or gene expression profiles at the whole embryonic level. Here, by data mining approach utilizing multiple genomic and transcriptomic datasets from different species in combination, and by experimental validation, we demonstrate that the hourglass model is also applicable to a reduced element, the spinal cord. In the middle of spinal cord development, dorsoventrally arrayed neuronal progenitor domains are established, which are conserved among vertebrates. By comparing the publicly available single-cell transcriptome datasets of mice and zebrafish, we found that ventral subpopulations of post-mitotic spinal neurons display divergent molecular profiles. We also detected the non-conservation of cis-regulatory elements located around the progenitor fate determinants, indicating that the cis-regulatory elements contributing to the progenitor specification are evolvable. These results demonstrate that, despite the conservation of the progenitor domains, the processes before and after the progenitor domain specification diverged. This study will be helpful to understand the molecular basis of the developmental hourglass model., (© 2021 The Authors. Development, Growth & Differentiation published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Developmental Biologists.)

Infections of CTX-M extended-spectrum β-lactamase-producing Enterobacterales are a severe threat in clinical settings. CTX-M genes on plasmids have been transferred to many Enterobacterales species, and these species have spread, leading to the global problem of antimicrobial resistance. Here, we developed a lateral flow immunoassay (LFIA) based on an anti-CTX-M rabbit monoclonal antibody. This antibody detected CTX-M variants from the CTX-M-9, CTX-M-2, and CTX-M-1 groups expressed in clinical isolates. The LFIA showed 100% sensitivity and specificity with clinical isolates on agar plates, and its limit of detection was 0.8 ng/mL recombinant CTX-M-14. The rabbit monoclonal antibody did not cross-react with bacteria producing other class A β-lactamases, including SHV. In conclusion, we developed a highly sensitive and specific LFIA capable of detecting CTX-M enzyme production in Enterobacterales. We anticipate that our LFIA will become a point-of-care test enabling rapid detection of CTX-M in hospital and community settings as well as a rapid environmental test., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Ikarugamycin (IK) is an antibiotic which has been reported to have a variety of functions, such as inhibition of clathrin-mediated endocytosis (CME), anti-tumor effects and regulation of the immune system. Whether IK influences cytokine production is poorly understood. We have investigated the relationship between IK and production of tumor necrosis factor-α (TNF). TNF plays a pivotal role in pathogenesis of many diseases. Although the dynamics of soluble TNF (sTNF) has been widely explored so far, the functions of the membrane form of TNF (mTNF) have not been fully elucidated. We demonstrated that IK increases the amount of mTNF and prolongs the duration of TNF expression. This effect is unrelated to the shedding activity of disintegrin and metalloproteinase domain-containing protein 17 (ADAM 17). Our results revealed that there is a mechanism to terminate inflammation at the cellular level which IK dysregulates. Furthermore, IK can be a tool to study TNF signaling due to its effect of increasing mTNF expression., Competing Interests: The authors have declared that no competing interests exist., (© 2021 The Author(s).)

Shiga toxin (STx) or Vero toxin is a virulence factor produced by enterohemorrhagic Escherichia coli . The toxin binds to the glycosphingolipid globotriaosylceramide (Gb3) for its entry, and causes cell death by inhibiting ribosome function. Previously, we performed a loss-of-function screen in HeLa cells using a human CRISPR knockout (KO) library and identified various host genes required for STx-induced cell death. To determine whether this library targeted to the human genome is applicable to non-human primate cells and to identify previously unrecognized factors crucial for STx-induced cell death, we herein performed a similar screen in the African green monkey kidney-derived Vero C1008 subline. Many genes relevant to metabolic enzymes and membrane trafficking were enriched, although the number of enriched genes was less than that obtained in the screening for HeLa cells. Of note, several genes that had not been enriched in the previous screening were enriched: one of these genes was SYS1 , which encodes a multi-spanning membrane protein in the Golgi apparatus. In SYS1 KO Vero cells, expression of Gb3 and sphingomyelin was decreased, while that of glucosylceramide and lactosylceramide was increased. In addition, loss of SYS1 inhibited the biosynthesis of protein glycans, deformed the Golgi apparatus, and perturbed the localization of trans -Golgi network protein (TGN) 46. These results indicate that the human CRISPR KO library is applicable to Vero cell lines, and SYS1 has a widespread effect on glycan biosynthesis via regulation of intra-Golgi and endosome-TGN retrograde transports.

Scabies is a highly contagious skin disease caused by the mite Sarcoptes scabiei that affects many mammals. However, the sensitivity of traditional tests for scabies diagnosis in humans is less than 50%. To simplify the diagnosis of scabies, methods that are simple, sensitive, specific, and cost-effective are required. We developed an immunodiagnostic test based on S. scabiei var. nyctereutis RNA-seq data collected from Japanese raccoon dogs with sarcoptic mange. Three candidate antigens-a highly expressed hypothetical protein "QR98&#95;0091190," another mite allergen known as "SMIPP-Cc," and an abundant "vitellogenin-like protein"-were evaluated by western-blot analysis. A lateral flow immunoassay, using specific antibodies against the vitellogenin-like protein, successfully detected scabies in the skin flakes of S. scabiei-infected raccoon dogs. This assay can potentially diagnose scabies more accurately in wildlife, as well as in humans.